Rapid progress has been made by many investigators trying to map the murine genome; consequently, the information and tools for genetic linkage studies in the mouse are expanding and readily available. There are several clear benefits to pursuing studies of human genetic disease in inbred mouse models. One advantage is that maximum genetic uniformity can be achieved in inbred progenitor strains and the F1 generation. Furthermore, F2 and backcross segregating generations can be easily produced. Secondly, environmental factors such as climate, air quality, light, and diet that can influence the phenotypic expression of many traits can be largely controlled in laboratory animal housing situations. Based on the observation of wide variation in AHR induced by intravenous acetylcholine challenge between different inbred strains of mice (4), we have chosen to investigate the etiology of this phenotype in a mouse model. Studies were initially performed in a naive murine model of AHR in which the A/J inbred mouse strain consistently demonstrated AHR to cholinergic challenge, and also in the C3H/HeJ strain, which was comparatively hyporesponsive to the same challenge (2, 4, 5). By studying segregating generations that derive from genetically hyperresponsive and hyporesponsive progenitor strains, we have demonstrated that a finite number of genes contribute to AHR in mice (2, 4, 5), indicating that pursuit of the genomic location of these genes was reasonable.

We performed studies designed to identify regions of DNA that were coinherited with the AHR phenotype in mice, as these coinherited regions would be likely to contain genes that determine AHR. The presence (or absence) of coinheritance between AHR and alleles at multiple DNA markers was determined in segregating backcross progeny ([C3H/HeJ × A/J] × A/J) of hyperresponsive (A/J) and hyporesponsive (C3H/HeJ) mice. Airway responsiveness was assessed by airway resistance, airway compliance, and airway pressure–time index (APTI) following intravenous acetylcholine challenge in mice receiving no other interventions. These measurements were made in anesthetized, mechanically ventilated mice of the A/J and C3H/HeJ strains and crosses derived from them: (C3H/ HeJ × A/J) F1, [(C3H/HeJ × A/J) F1 × (C3H/HeJ × A/J) F1] F2, [(C3H/HeJ × A/J) F1 × A/J] A/J backcross, and [(C3H/HeJ × A/J) F1 × C3H/HeJ)] C3H/HeJ backcross (Figure 1). After phenotyping for airway responsiveness, DNA was isolated from tissues collected from the A/J backcross population. Simple sequence repeat DNA markers (n = 94) that varied between the A/J and C3H/HeJ strains were amplified by polymerase chain reaction (PCR) using A/J backcross template DNA. Markers surrounded candidate genes or were positioned at approximately 20 cM intervals throughout the genome. Airway hyperresponsiveness genes were then mapped via quantitative trait locus (QTL) linkage analysis using the MAPMAKER/QTL statistical package (generously provided by S. E. Lincoln, M. J. Daly, and E. S. Lander of the Whitehead Institute for Biomedical Research, Cambridge, Massachusetts).

Fig. 1. Frequency distribution of APTI induced by 50 μg/kg acetylcholine given intravenously to A/J (n = 58) and C3H/HeJ (n = 53) mice and the progeny of crosses between them. F1 (n = 25) are the offspring of crosses between female C3H/HeJ and male A/J mice. F2 (n = 60) are the offspring of the intercross between F1 mice. A/J backcross (n = 196) are the offspring of the backcross between female F1 and male A/J mice. C3H/HeJ backcross (n = 15) are the offspring of the backcross between female F1 and male C3H/HeJ mice. Power transformation (p) of the original data (y) are presented (x) where x = y^p, p = –0.1498. (Results are from Reference 2.)

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A QTL for AHR was detected on murine chromosome 6. A maximum lod score of 3.11 for APTI was achieved on the midportion of chromosome 6 between makers D6Mit62 and D6Mit13, 2 cM distal to marker D6Mit62. Alleles at the marker D6Mit62 had significant effect on the AHR phenotype (Figure 2). The A alleles were derived from the A/J progenitors and H alleles were from C3H/HeJ progenitors. The A allele homozygous state in both A/J and A/J backcross mice was associated with an approximately threefold greater airway response than the heterozygous condition. The H allele was correlated with minimal airway responsiveness in C3H/HeJ mice and in heterozygous A/J backcross mice.

Fig. 2. Effect of the genotype at linked locus D6Mit62 on the acetylcholine-induced APTI in A/J, C3H/HeJ, and A/J backcross mice. A/A mice are homozygous for A/J alleles, A/H mice are heterozygous, and H/H mice are homozygous for C3H/HeJ alleles at D6Mit62. *Denotes significant allelic effect on the trait (p < 0.05). A/J backcross mice genotyped are in the hyporesponsive and hyperresponsive tails of the population distribution. (Results are from Reference 2.)

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De Sanctis and co-workers found AHR to be a polygenic trait in a similar murine model (3). They studied the inheritance of the naive AHR response to methacholine challenge in crosses between hyperresponsive A/J mice and hyporesponsive C57BL/6 mice. Linkage analysis of the C57BL/6 backcross generation yielded three QTLs located on murine chromosome 2 (lod = 3.0 at D2Mit409), chromosome 15 (lod = 3.7 on D15Mit107), and chromosome 17 (lod = 2.83 on D17Mit26). Differences in map locations of naive AHR genes in these two models may be due to several factors, including differences in hyporesponsive strains studied (C3H/HeJ-v-C57BL/6) and differences in methods of AHR assessment (2, 3, 6).

The gene encoding the interleukin (IL)-5 receptor-α maps to chromosome 6 (47 cM from the centromere) between the markers linked to naive AHR, D6Mit104 and D6Mit10. Interleukin-5 is likely to be important in allergic responses because it promotes growth and differentiation of B cells, stimulates IgA synthesis, and induces differentiation and activation of eosinophils. Based on studies of human asthma in which the IL-5 gene is linked to bronchial hyperresponsiveness (1), the IL-5 receptor may logically be in the mechanistic pathway that produces this phenotype in the mouse. Additionally, CD4, CD8a, and CD8b antigens, present on the cell surface of helper and suppressor/cytotoxic T cells, respectively, are encoded on mouse chromosome 6 within the confidence interval surrounding the putative QTL (2). The map location on chromosome 6 of a QTL that contributes to AHR in A/J and C3H/ HeJ mice under noninflammatory conditions provides a foundation for further studies regarding the map location, and ultimately the identification, of gene(s) that determine this phenotype in antigen-treated mice with airway phenotypes that closely parallel atopic asthma.

In an attempt to replicate more closely the eosinophilic airway inflammation of human asthma, we developed a murine model of AHR in which antigen-treated mice develop airway inflammation and eosinophilia (7). Based on the magnitude of the inherent airway contractile responses to acetylcholine stimulation in the A/J and C3H/HeJ strains under naive (noninflammatory) conditions, we selected these two strains for further study under antigen-treated conditions. Mice were sensitized and challenged with antigen, either sheep red blood cells (SRBC) or ovalbumin (OA), by similar protocols (7, 8). Mice were injected intraperitoneally with antigen in phosphate-buffered saline (PBS). Sham-sensitized mice received an equal volume of PBS, while other mice remained untreated. After 14 to 17 d, antigen-sensitized and sham-sensitized animals were challenged intratracheally under anesthesia with the sensitizing antigen. Sham-challenged mice received an equal volume of PBS without antigen, and no intervention was made in the untreated mice. After 3 d, airway responsiveness to acetylcholine was measured, as in the naive model. Other analyses performed include cytology of bronchoalveolar lavage (BAL) fluid, lung histology, flow cytometry, cytokine bioassays of BAL fluid, and cytokine mRNA analysis of lung tissue.

After antigen sensitization and challenge, A/J mice demonstrated an increase in airway responsiveness approximately twofold their response under naive conditions (Figure 3). Along with this exaggerated airway contractile response, antigen treatment also induced airway inflammation in A/J mice, characterized by increased numbers of macrophages, neutrophils, lymphocytes, and eosinophils in BAL fluid. Most striking were the increases in lavaged lymphocytes and eosinophils. Lungs from antigen-treated mice contained large numbers of perivascular and peribronchial mononuclear cells and eosinophils, along with a large number of goblet cells.

Fig. 3. Effect of various sensitization and challenge protocols on acetylcholine-induced airway responsiveness in A/J mice. Mice were untreated or were sensitized and challenged with SRBC antigen or PBS vehicle (sham) as indicated. Data are means and SE of 5–19 mice per group. *p < 0.05 compared to all other groups. (Results are from Reference 7.)

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The C3H/HeJ strain reacted to antigen treatment with a vigorous lung inflammatory response characterized by increased numbers of lavaged alveolar macrophages, neutrophils, lymphocytes, and eosinophils as compared to control animals (9). The number of macrophages and neutrophils was significantly greater in antigen-treated C3H/HeJ mice than in antigen-treated A/J mice. Despite the somewhat greater inflammatory response in the C3H/HeJ strain, those mice remained hyporesponsive to cholinergic-induced bronchoconstriction (9). Thus, the A/J strain demonstrated AHR to cholinergic stimulation in the absence of antigen exposure or airway inflammation and augmented AHR in the presence of antigen treatment, while the C3H/HeJ strain was hyporesponsive in both the naive and antigen-induced inflammatory states.

In this model we were able to simulate in mice several of the hallmarks of human asthma including AHR, pulmonary inflammation characterized by eosinophilia, and goblet cell hyperplasia. Furthermore, we have identified inbred strains of mice that are genetically predisposed to be susceptible (A/J) or resistant (C3H/HeJ) to the bronchoconstrictor effects of cholinergic agonists under inflammatory and noninflammatory conditions.

Because T lymphocytes orchestrate specific immune response to foreign antigens, considerable interest exists in their potential role in the inflammatory responses observed in the lungs of allergic asthmatics. Definitive proof of the role of T cells in humans is difficult to obtain; therefore, animal models, particularly murine models, have been extremely useful for delineating the role of both T cells and T-cell–derived cytokines in the development of AHR. In this regard, we examined the importance of T helper cells in AHR and eosinophilia from studies in which antigen-sensitized A/J mice were treated with rat antimouse CD4+ monoclonal antibody, GK1.5, before antigen challenge (7). Mice were injected intraperitoneally with GK1.5 or control rat IgG 3 d before intratracheal antigen challenge. Depletion of CD4+ T cells was confirmed by flow cytometry of mouse spleen cells. After depletion of CD4+ T cells, the intratracheal antigen challenge was delivered. The AHR was measured and BAL fluid cytology and lung histopathology were performed as previously described (7). Depletion of CD4+ T cells significantly inhibited the increased airway responsiveness induced by antigen treatment in the mice not receiving GK1.5. A significant reduction in the numbers of antigen-induced BAL fluid macrophages, neutrophils, lymphocytes, and eosinophils was noted in the mice with CD4+ T-cell depletion. The GK1.5 treatment before antigen challenge also significantly reduced the numbers of all cell types in the pulmonary interstitial cell compartment as compared to mice receiving antigen but not GK1.5 treatment. Treatment with GK1.5 had no effect on cell populations in the BAL fluid or pulmonary interstitium in sham-treated mice. The ablation of antigen-induced AHR and cell infiltration by T helper cell depletion provides conclusive evidence that CD4+ lymphocytes mediate antigen-induced AHR and pulmonary cellular infiltration. Subsequently, these studies have been confirmed by other groups of investigators (10).

At least two subsets of T helper (CD4+) cells, Th1 and Th2, have been identified in mice. These subsets can only be differentiated on the basis of the cytokine profiles that they produce when stimulated (11). Knowledge of the precise cytokine profiles elaborated by each of these subsets is evolving, but it is widely accepted that Th1 cells produce IL-2, interferon-γ (IFN-γ), and tumor necrosis factor-β (TNF-β), while Th2 cells produce IL-4, IL-5, IL-10, and IL-13 (12). Both subsets of murine T helper cells produce IL-3 and granulocyte/macrophage colony-stimulating factor (GMCSF). The relevance of these T helper subsets in humans is less clear, as there is greater overlap in the cytokines produced by both Th1 and Th2 cells. In humans, the unique cytokines are limited to IFN-γ and TNF-β for Th1 cells and IL-4 and IL-5 for Th2 cells (12).

The Th2 cytokines are important for immune regulation, and their specific functions make them ideal candidates for the study of antigen-associated AHR and inflammation. In particular, IL-4 may be of critical importance in allergic inflammatory processes, as it is required for the differentiation and expansion of Th2 lymphocytes. Furthermore, IL-4 inhibits the development of Th1 lymphocytes. Interleukin-4 is a B cell growth factor; it enhances the antigen-presenting capacity of B cells, induces immunoglobulin isotype switching to IgE, and is also a T cell growth factor. The genes encoding IL-4 and the IL-4 receptor-α map to murine chromosomes 11 (20 cM from the centromere) and 7 (62 cM from the centromere), respectively. The function of other Th2 cytokines has also made them popular candidates for involvement in allergen-mediated hypersensitivities. Interleukin-13 is a recently identified cytokine that is very similar to IL-4 in regard to both sequence homology and effects on B cell activity and IgE class switching (13). In contrast to IL-4, IL-13 is not involved in the differential development of Th1 and Th2 cell populations. Interleukin-5 is produced by mast cells and eosinophils as well as Th2 cells. It functions primarily as a chemotaxin and activator of eosinophils. Interleukin-5 induces eosinophil degranulation and prolongs survival by limiting apoptosis of eosinophils. Interleukin-10, produced by murine Th2 cells, downregulates the synthesis of IL-2, IL-4, and other pro-inflammatory cytokines. Deficiency of this anti-inflammatory cytokine may permit inflammatory diseases such as asthma (14). Based on the actions of these Th2 cytokines, there has been much interest regarding their potential role in allergic diseases, including atopy and asthma.

The ontogeny of Th1 and Th2 subsets remains unclear. The data, from limiting dilution analysis (15) and studies using T-cell receptor transgenic mice (16), suggest that naive T helper cells have the ability to differentiate into either Th1 or Th2 cells. Naive T cells (Th0) generate IL-2 when activated by antigen presentation and costimulatory signals, then progress through a multipotential cell that generates a mixed spectrum of cytokines, including IL-2, IL-4, and IFN-γ. Although many immune responses likely retain the Th0 phenotype, responses may be polarized to either a Th1 or Th2 type, a process that is markedly influenced by the nature of the antigen, the dose of antigen, and the cytokine milieu during initial priming by specific antigens (17). The importance of altered lymphokine levels in the immunopathogenesis of disease is well documented. Skewed production of CD4+ T lymphokines has been shown to be important in the pathogenesis of several diseases such as Lyme disease, rheumatoid arthritis, measles, and other infectious and parasitic diseases (18, 19).

Perhaps due to their highly inbred nature, several mouse strains appear to have a genetic predisposition to either Th1 or Th2 responses, independent of the provocation. Both C57BL/6 and C3H/HeJ mice tend to mount Th1 immune responses, while BALB/c and A/J mice are predisposed to Th2 responses. Because of their “predetermined” immune response, these mice are inherently resistant or susceptible to the effects of select immunostimuli based on whether their inherent response is “correct.” For example, clearance of Leishmania major infection is Th1-dependent; therefore, C57BL/6 mice are resistant and BALB/c mice are susceptible to this infection (20).

A possible immunopathogenic role for Th2 CD4+ T cells in allergen-induced AHR is suggested by several lines of evidence. Firstly, elevations in Th2 cytokines have been consistently observed in the lungs of human asthmatics (21-23) and allergen-challenged animals (7, 24). Further evidence to support this hypothesis includes our findings that A/J mice, which inherently mount a Th2 response, demonstrate antigen-induced airway hypersensitivity that is similar to asthma, while C3H/ HeJ mice, which mount a Th1 response, are relatively protected against the antigen-induced hypersensitivity phenotype (9). Secondly, studies in which IL-4 and IL-5 have been manipulated either through antibody blockade or through gene targeting have shown that these cytokines are essential to the development of allergic inflammation and AHR (24-26). For example, we have studied the role of Th2 cytokines in antigen-induced AHR and inflammation in susceptible A/J mice (25). Interleukin-4 involvement in susceptibility to antigen- induced AHR in A/J mice was examined by blocking the IL-4 receptor via administration of anti–IL-4R monoclonal antibody (mAb), M1, in sensitized mice 3 d before intratracheal antigen challenge. Cholinergic-induced AHR was abrogated following IL-4R blockade (Figure 4). Antigen-induced increases in pulmonary goblet cells and eosinophils were also blocked by anti–IL-4R mAb. Cytokine profiles were measured by reverse transcriptase polymerase chain reaction (RT-PCR) in lungs of A/J mice after antigen treatment protocols (25). Untreated and sham-treated animals had very low levels of IL-4, IL-5, and IFN-γ mRNA. Their IL-4 and IL-5 levels were significantly increased after antigen sensitization and challenge, while levels of IFN-γ were unchanged. Treatment with anti–IL-4R did not affect antigen-induced increases in lung mRNA and BAL protein levels of IL-5 and IFN-γ or IL-4 mRNA.

Fig. 4. Effects of anti–IL-4 receptor mAb on acetylcholine-induced airway responsiveness in mice 3 d after a single intratracheal challenge with SRBC antigen. Values shown are means and SE of 7–10 mice per group. Sham or SRBC-sensitized mice were injected intraperitoneally with 3 mg anti–IL-4 receptor mAb (M1) or an isotype control mAb (GL117) 3 d before intratracheal challenge. *Denotes significant difference from other groups, p < 0.05. (Results are from Reference 25.)

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Further support that IL-4 is required for the development of antigen-induced AHR is provided by the finding that OA-induced AHR, increased serum IgE, and increased BAL eosinophil and lymphocyte numbers were inhibited in C57BL/6 IL-4–deficient mice as compared to IL-4 sufficient mice (26, 27). Several studies have shown that in vivo neutralization of IL-4 at the time of antigen immunization suppressed Ag- induced AHR (28) and eosinophilia (27); however, when anti– IL-4 mAb treatment was limited to the antigen challenge period, there was no effect on these parameters (27, 28). These studies suggest that IL-4 is required for priming of Th2 cells early in the response to antigen, but that once a Th2 pattern is established IL-4 is not required to maintain Th2 cytokine production. Evidence of this is provided by the observations that Th2 cytokine production is not inhibited in Ag-exposed animals receiving either anti–IL-4 mAb (27) or anti–IL-4 receptor mAb (25) before local antigen challenge. In addition to its primary role in T-cell differentiation, IL-4 regulates a number of processes potentially important in allergic inflammation, such as IgE production, mastocytosis, and upregulation of vascular cell adhesion molecule-1 (VCAM-1) expression on endothelium. The relative importance of each of these IL-4– mediated processes in the development of antigen-induced airway hyperresponsiveness is unclear at the present time.

One of the potential mechanism(s) by which IL-4 may induce AHR is its ability to upregulate endothelial VCAM-1 expression on endothelium. Interaction of VCAM-1 with the very late activation antigen-4 (VLA-4) promotes eosinophil recruitment (29). Therefore, we studied the effect of VCAM-1 and VLA-4 depletion on antigen-induced responsiveness and inflammation in our model (25). Antibodies to VCAM-1 and VLA-4 were administered to antigen-sensitized mice 1 d before antigen challenge. Although BAL eosinophils were reduced following VCAM-1 and VLA-4 blockade, there was no change in AHR or the number of macrophages and lymphocytes in BAL. These findings have not been supported consistently by two other studies (30, 31). Both of those found that blockade of VLA-4 inhibited antigen-induced late responses and hyperreactivity; only one showed it to reduce BAL eosinophil numbers. Thus, further work needs to be done before studies on VCAM-1 or VLA-4 blockade can shed any light on the role of IL-4 in eosinophilic inflammation.

Taken together, these studies demonstrate the importance of IL-4 in mediating inflammation and constriction of murine airways in response to antigen exposures. Due to the pleiotropic effects of IL-4 on specific immune responses to antigen exposure, the exact mechanisms of its actions remain to be defined. However, these studies suggest the possibility that genes important in the regulation of, production of, or response to IL-4 may underlie allergic asthma. Furthermore, the possibility exists that antagonism of IL-4 protein or receptors may limit the signs of asthma.

Interleukin-5 is a likely candidate in the development of AHR because it has been shown to be the primary determinant of eosinophil differentiation, activation, and survival. The gene encoding IL-5 maps to murine chromosome 11 (28 cM from the centromere). The relationship between IL-5 and eosinophilia is well-established in both guinea pig and mouse models of allergic inflammation. However, the relationship between eosinophilia and AHR is not as clear. For example, IL-5 transgenic mice exposed to OA show a marked eosinophilia, but do not develop AHR (32). On the other hand, prolonged delivery of recombinant IL-5 to naive mice has been shown to induce AHR concomitant with pulmonary eosinophilia and increased mucus production (33). Taken together these data suggest that other factors besides IL-5 may be required to induce AHR or that AHR can be induced by multiple mechanisms in the context of different genetic backgrounds.

Collectively, these studies support a role for the Th2 cytokines in the pathogenesis of allergen-induced AHR and eosinophilic inflammation. Further studies are needed to determine the mechanisms by which T cells and/or T-cell–derived cytokines induce alterations in the regulation of airway tone, as well as the mechanisms that drive the differentiation of Th2 cells in response to allergen exposure in susceptible animals.

The cytokines IL-4 and IL-12 are thought to be the primary regulators of Th2 and Th1 cell differentiation, respectively. Interleukin-4, as discussed above, induces the differentiation of CD4+ T cells into Th2 cells and is required for the expansion of Th2 cell clones, while inhibiting Th1 cell expansion. Interleukin-12, a product of macrophages and other antigen-presenting cells, is required for expansion of Th1 clones and suppresses Th2 cell production. Interleukin-12 is a heterodimer of 70 kD formed by two covalently linked glycosylated chains of approximately 40 kD (p40) and 35 kD (p35) (34). The gene encoding the p40 subunit is located on mouse chromosome 11 (17.5 cM from the centromere), homologous to human 5q, and the p35 subunit is located on chromosome 3 (37 cM from the murine centromere) in mice and humans. The p40 subunit is highly regulated by other cytokines, such as IFN-γ, TNF-β, IL-10, and the prostaglandin E2.

To pursue the role of T helper cell differentiation in determining the phenotypes of interest, investigations have focused on the roles of IL-4 and IL-12 in allergen-induced airway changes. Following the studies, described above, in which inhibition of IL-4 protein or receptor attenuated antigen-induced AHR and pulmonary inflammation, we have examined the effects of IL-12 on airway responses to antigen (35). A/J mice sensitized to antigen 2 wk earlier were treated with murine recombinant IL-12 (mrIL-12) at –2, –1, 1, and 2 d relative to intratracheal antigen challenge. Additional mice were given two intratracheal antigen challenges 7 d apart, and mrIL-12 was administered –2, –1, 1, and 2 d relative to the second antigen challenge. Supplemental IL-12 reduced in a dose-dependent manner the AHR associated with a single challenge of antigen (Figure 5). Pulmonary eosinophils, lymphocytes, and neutrophils were reduced by mrIL-12 as compared to antigen-treated control animals. Antigen-induced elevation in lung mRNA for IL-4 and IL-5 was abolished following IL-12 treatment. Pulmonary IFN-γ mRNA and protein, which were low before and after antigen exposure, were increased by IL-12 treatment (35). Others report that mrIL-12 treatment in C57BL/6 mice reduced the antigen-induced influx of eosinophils in BAL fluid, inhibited IgE synthesis, and abolished antigen-induced AHR (10). We also found that IL-12 given once an inflammatory response was established in the lung inhibited antigen-induced airway contractility and inflammatory responses (35).

Fig. 5. Dependence of IL-12 effects on IFN-γ. Antigen-challenged mice were injected intraperitoneally with vehicle or 1 μg IL-12 for 5 d beginning 2 d before antigen challenge. Measurements were determined 3 d after challenge. Some groups were injected intraperitoneally with 2 mg isotype-matched control mAb or anti–IFN-γ mAb at –2 d and +1 d relative to challenge. Results shown are means and SE of 3–6 mice per group. *p < 0.05 compared with antigen-challenged untreated groups and group treated with IL-12 and anti–IFN-γ; ^†p = 0.05 compared with all except antigen-only group; ^§p = 0.05 compared with all except antigen + isotype and antigen + anti–IFN-γ groups. (Results are from Reference 35.)

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Measurement of cytokine levels in lungs of mice treated with both antigen and mrIL-12 suggested that the effects of IL-12 may be mediated through IFN-γ release. To further assess this idea, mrIL-12 was administered concurrently with antibody to IFN-γ. Anti–IFN-γ mAb partially reversed the IL-12–induced inhibition of AHR and pulmonary eosinophilia in antigen-treated mice (35) (Figure 5). Thus, the effects of IL-12 are dependent, at least in part, on the presence of IFN-γ. These studies are consistent with other studies showing the effectiveness of both aerosol (36) and gene delivery of IFN-γ (37) in inhibiting the development of allergic airway responses to antigens in mice.

These studies demonstrate that the AHR and pulmonary inflammation induced by antigen challenge are associated with a Th2 pattern of cytokine expression in the murine lung. Interleukin-12 administration prevented the development of these responses and suppressed the antigen-induced increases in expression of the Th2 cytokines, IL-4 and IL-5. The effects of IL-12 were reduced in the absence of IFN-γ, another essential Th1 cytokine. Furthermore, IL-12 is effective in altering the airway phenotype in the presence of fully established airway inflammation induced by repeated antigen challenge. This supports the idea that local administration of IL-12, or agents that induce production of IL-12, may be useful in the treatment of chronic allergic conditions such as atopic asthma.

The ability of exogenously added IL-12 to ablate allergic airway responses in A/J mice suggests that endogenous levels of IL-12 may be low or that the cellular responses to IL-12 are abnormal in susceptible A/J mice. This hypothesis is supported by the fact that A/J mice are also susceptible to Listeria infection, in which IL-12 is required for successful resolution of the infection. Alternatively, we might predict that resistant strains of mice, such as C3H/HeJ mice, may produce sufficient amounts of IL-12 in response to local lung provocation with allergens, thus suppressing the development of aberrant Th2 cytokine patterns and subsequent AHR. Clearly, further studies are needed to explore the possible role of dysregulation of the immunoregulator cytokine, IL-12, in susceptibility to allergic airway disease.

Recently, it has become appreciated that CD4+ T-cell activation leading to cytokine production and effector function requires two signals from antigen-presenting cells (APCs) (38). The first signal is triggered by interaction of the antigen-specific T-cell receptor (TcR) with peptide–major histocompatibility complex (MHC) II complexes on APCs, while the second costimulatory signal is not antigen-specific. One of the most well-characterized costimulatory signaling pathways involves the CD28 and cytolytic T lymphocyte–associated antigen (CTLA)-4 molecules, which are expressed on T cells, and their ligands, B7-1 (CD80) and B7-2 (CD86), which are expressed on APCs (39). The gene encoding CD80 is located on mouse chromosome 16 (25.1 cM from the centromere). Interaction with CD28 on T cells provides a second signal, inducing T cells to proliferate and produce IL-2. Failure to receive this second signal can result in anergy (40). In contrast, the second receptor for B7, CTLA-4, appears to mediate a negative signal. The gene location of murine CTLA-4 is chromosome 1 (30.1 cM from the centromere). Blockade of the B7/CD28– CTLA-4 pathway with CTLA-4-Ig, a soluble fusion protein, has been shown to effectively inhibit T-cell activation in vitro and in vivo (41). Additionally, studies suggest that B7/CD28– CTLA-4 interactions may not only be important in T-cell activation and IL-2 production, but they may also play a role in T-cell differentiation into Th1 and Th2 cells (42). As susceptible A/J and resistant C3H/HeJ mice appear to differ in their immune response to allergen challenge, we were interested in determining whether B7/CD28–CTLA-4 interactions may be important in the airway responses of A/J mice to OA provocation. The A/J mice were given CTLA-4-Ig or the control fusion protein, L6, either before antigen sensitization or before pulmonary antigen challenge (8). Interestingly, CTLA-4-Ig administration significantly suppressed antigen-induced AHR whenever administered (Figure 6). Concomitant with the ablation of AHR, CTLA-4-Ig treatment suppressed BAL eosinophil numbers, suppressed antigen-induced increases in IL-4, and significantly suppressed antigen-induced increases in total serum IgE levels. These results suggest that B7/CD28–CTLA-4 costimulation is required for the development of many of the physiologic features of allergic asthma in this model, possibly by promoting a pathologic type 2 associated immune response. As expression of these costimulatory molecules on APCs is known to vary, differential expression of these molecules may lead to either development of tolerance or progression of pathologic immune responses in response to allergen exposure. Further studies will be needed to evaluate the role of these molecules in the resistance to the development of AHR in C3H/HeJ mice.

Fig. 6. CTLA-4-Ig administration (total 200 μg/mouse) inhibits OA-induced increases in airway reactivity to intravenous acetylcholine challenge (50 μg/kg). Mice were injected with L6 the control fusion protein or CTLA-4-Ig either (A) before OA or PBS sensitization (10 μg OA in 200 μl of PBS or an equivalent volume of PBS alone); or (B) before local challenge with either OA or PBS (50 μl of 1.5% solution of OA or equivalent volume of PBS). Values shown are mean ± SE of mice per group. *Denotes those values significantly different from those of OA groups, p < 0.05; ⁺denotes those values significantly different from those of PBS groups. (Results are from Reference 8.)

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The results of these studies provide substantial insight into mechanisms that may underlie antigen-induced AHR and pulmonary inflammation. The substantial database on the immunological aspects of antigen-induced airway phenotypes in A/J and C3H/HeJ mice, along with the information regarding the genetic basis of the noninflammatory airway phenotype in these strains, provide a tremendous tool for identifying the genes that determine these antigen-induced airway changes. We have additional preliminary evidence to suggest that the gene(s) determining antigen-induced AHR and pulmonary inflammation may be unique to the genes that determine naive AHR in these strains. Future studies will aim to determine the specific mode of inheritance for antigen-induced AHR and identify the responsible gene(s).